Sunday, 25 April 2021

HAT Medium : Principle & Selection

 HAT stands for Hypoxanthine Aminopterin Thymidine. The Hypoxanthine and Thymidine are the bases which are converted into nucleotides by the enzymes HGPRTase and Thymidine kinase respectively.
•  The pathway of nucleotide synthesis, which involves these two key enzymes is known as the salvage pathway
•  Aminopterin is an inhibitor of the de novo pathway of nucleotide synthesis.

HAT Medium

  Mammalian cells can be isolated from their body. These cells can be grown in cell cultures that is under suitable artificial conditions.
  HAT medium is a selection medium for mammalian cell culture.
  Suppose we have two type of mammalian cells.
1) First is the antibody producing. These cells have a limited lifespan in cell culture.
2). Second cell type are immortal b-cells. These cells are capable of dividing indefinitely in cell culture.
  Another important thing to note is that these cells are mutant for the genes producing the enzyme HGPRT and antibodies. So we will represent these feature as HGPRT negative and IG negative.
This means these cells cannot synthesize nucleotide by the salvage pathway.
They cannot produce their own antibodies.

We mix these two type of cells and expose them to polyethylene glycol(PEG). PEG is chemical fusogen.
In PEG medium the cells will fuse with each other. As a result of this fusion we will have five type of cells in the mixture -
1). Unfused antibody producing b-cell
2). Unfused immortal b-cell
3). Fused antibody producing b-cells
4). Fused immortal b-cells and
5). Hybrid cells formed by fusion of antibody producing b-cell and immortal b-cell.

Five types of cells after fusion process

Separation of Hybrid cells

  Our next aim is to separate these hybrid cells from this mixture of cells. This cell mixture is now transferred to the HAT medium.

• The antibody producing cells have a short lifespan in cell culture.
• So after some time the unfused and fused antibody producing B-cells will die.
• Immortal b-cells (HGPRT negative) cannot synthesize nucleotides by the salvage pathway.
• Also this HAT medium contains Aminopterin which blocks the de novo pathway of nucleotide synthesis.
• Since both the pathways of nucleotide synthesis are blocked. the unfused and fused immortal b-cells will also die.
• The only remains is the hybrid cells formed by the fusion of antibody producing b-cell and immortal b-cell.
• These hybrid cells will survive in the HAT medium. This is because they are able to divide indefinitely.
- This property is due to the immortal B-cell partner.
- Functional HGPRT enzyme for nucleotide synthesis is provided by the antibody producing b-cell partner.
So these cells will be able to synthesize nucleotides by the salvage pathway.
HAT medium has the function of artificial selection in cell culture techniques. This medium selects those cells which have functional HGPRT and TK enzymes. 

 HAT medium is mainly used in Hybridoma Technology for production of monoclonal antibodies.


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